Hpts-mono and bis cys-ma polymerizable fluorescent dyes for use in analyte sensors

ABSTRACT

Novel fluorescent dyes are disclosed for use in analyte detection. In particular, mono- and bis-substituted HPTS dyes and methods of making them are provided.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/954,204 filed Aug. 6, 2007, which is hereby incorporated by referencein its entirety.

FIELD OF THE INVENTION

Novel fluorescent dyes are disclosed for use in analyte detection.

DESCRIPTION OF THE RELATED ART

Investigators have used fluorescent techniques to measure polyhydroxylcompound (e.g., glucose) concentrations in body fluids. For example,Russell, disclosed the use of a boronic acid fimctionalized dye thatbinds to glucose and generates a signal dependent on glucoseconcentration (U.S. Pat. No. 5,512,246). James et al. used the sameprinciple but combined a fluorescent dye, an amine quenchingfunctionality, and a boronic acid in a single complex moiety, thefluorescence emission from which varies with the extent of glucosebinding (U.S. Pat. No. 5,503,770). Glucose sensors comprising afluorescent dye and a quencher comprising a single viologen moietyappended with boronic acids have been synthesized and investigated(e.g., Gamsey, S. et al. 2006 Langmuir 22:9067-9074; Thoniyot, P. et al.2006 Diabetes Technol Ther 8:279-287; Cordes, D. B. et al. 2005 Langmuir21:6540-6547; Cordes, D. B. et al. 2005 Org Biomol Chem 3:1708-1713;Cappuccio, E. E. et al. 2004 J Fluoresc 14:521-533; Gamsey, S. et al.2007 J Am Chem Soc 129:1278-1286 and Cordes, D. B. et al. 2006 AngewChem Int Ed Engl 45:3829-3832).

Fluorescent dyes, including 8-hydroxypyrene-1,3,6-trisulfonic acid(HPTS) and its derivatives, are known and have been used in analytedetection. See e.g., U.S. Pat. Nos. 6,653,141, 6,627,177, 5,512,246,5,137,833, 6,800,451, 6,794,195, 6,804,544, 6,002,954, 6,319,540,6,766,183, 5,503,770, and 5,763,238; International Application No.PCT/US2003/030167; and co-pending U.S. patent application Nos.10/456,895 and 11/296,898; each of which are incorporated by referencein their entireties. Although International Application No.PCT/US2003/030167 describes bis-substituted HPTS derivatives, they arestructurally different from the bis-substituted HPTS compounds disclosedherein and the synthesis methods described are different from themethods disclosed herein.

Segue to the Invention

As part of an ongoing effort to synthesize analyte sensors, we havedeveloped new mono- and bis-substituted HTPS fluorescent dyes. Thesedyes may be used in combination with analyte-binding moieties to achievereal-time measurement of analyte levels in vivo.

SUMMARY OF THE INVENTION Mono-Substituted Dyes

N-substituted mono-sulfonamide derivatives of HPTS having the genericstructure below are disclosed in the present invention:

-   -   wherein R¹ and R² are independently selected from the group        consisting of H, an anionic group and a reactive group, with the        proviso that R¹ and R² collectively comprise at least one        anionic group and at least one reactive group; and wherein M is        a counterion.

In some embodiments, the anionic group is sulfonic acid.

In some embodiments, the reactive group is an ethylenically unsaturatedpolymerizable group selected from the group consisting of acryloyl,methacryloyl, acrylamide, methacrylamido, styryl, and the like.

In some embodiments, the reactive group comprises a coupling groupselected from the group consisting of a carboxylic acid, aldehyde,alkyne, azide, activated ester, succinimide and nitrobenzoate, andwherein the coupling group is capable of binding the compound to apolymer or substrate.

In embodiments wherein one of R¹ and R² is H, the other group includesboth an anionic group and a reactive group.

In some embodiments, R¹ and R² are bonded together in a cyclicstructure.

-   1. Mono-CysMA

A mono-substituted fluorescent dye termed mono-CysMA having thestructure below is disclosed in accordance with preferred embodiments ofthe present invention.

A method of making mono-CysMA is disclosed in accordance with anotherembodiment of the present invention. The method comprises the steps of:

-   2. Mono-MA

Another mono-substituted fluorescent dye termed mono-MA having thestructure below is disclosed in accordance with preferred embodiments ofthe present invention.

A method of making mono-MA is disclosed in accordance with anotherembodiment of the present invention. The method comprises the steps of:

Bis-Substituted Dyes

N-substituted bis-sulfonamide derivatives of HPTS having the genericstructure below are disclosed:

-   -   wherein R¹ and R² are independently selected from the group        consisting of H, an anionic group and a reactive group, with the        proviso that R¹ and R² collectively comprise at least one        anionic group and at least one reactive group; and wherein M is        a counterion.

In some embodiments, the anionic group is sulfonic acid.

In some embodiments, the reactive group is an ethylenically unsaturatedpolymerizable group selected from the group consisting of acryloyl,methacryloyl, acrylamide, methacrylamido, styryl, and the like.

In some embodiments, the reactive group comprises a coupling groupselected from the group consisting of a carboxylic acid, aldehyde,alkyne, azide, activated ester, succinimide and nitrobenzoate, andwherein the coupling group is capable of binding the compound to apolymer or substrate.

In some embodiments, R¹ and R² are bonded together in a cyclicstructure.

A bis-substituted fluorescent dye termed bis-CysMA having the structurebelow is disclosed in accordance with preferred embodiments of thepresent invention.

A method of making bis-substituted dyes is disclosed in accordance withanother embodiment of the present invention. The method comprises thesteps of:

Glucose Sensors

A glucose sensor is disclosed in accordance with another embodiment ofthe present invention, comprising a mono- or bis-substituted dyedescribed herein and a quencher comprising boronic acid such as boronicacid-substituted viologens, or pyridinium and quinolinium saltsfunctionalized with boronic acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Comparison of mono-CysMA with tri-CysMA in 40% DMAA at pH 5.

FIG. 2. pH profile of mono-CysMA.

FIG. 3. Mono-CysMA response to glucose (mono-CysMA+3,3′-oBBV in 40%DMAA).

FIG. 4. Fluorescence spectra of mono-MA at different pH.

FIG. 5. Fluorescence spectra of mono-CysMA at different pH.

FIG. 6. Fluorescence spectra of tri-CysMA at different pH.

FIG. 7. Stem-Volmer comparison of HPTS dyes with 3,3′-oBBV.

FIG. 8. Comparison of glucose response with different dyes and3,3′-oBBV.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Dyes

As used herein, the terms “fluorophore” or “fluorophore dye” or “dye”refer to a compound that, when exposed to light of appropriatewavelength, emits light, i.e., it fluoresces.

As used herein, a “coupling group” is a reactive functional group,capable of forming a covalent bond with a polymer, substrate matrix etc.especially with a preformed hydrogel. Such groups include, but are notlimited to carboxylic acids, aldehydes, alkynes and azides, as well asactivated esters, such as succinimides and nitrobenzoates, or any othermonofimctional linker chemistry capable of covalently binding withpolymer, substrate matrix etc. especially with a preformed hydrogel.

As used herein, an “anionic group” is any negatively charged group(e.g., SO₃ ⁻, HPO₃ ⁻, CO₂ ⁻ and

As used herein, a “counterion” is an ion that associates with an ion ofopposite charge in the dye molecule. Non-limiting example counterionsinclude H⁺, an alkali metal ion, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, Fr⁺, an oniumion and NR₄ ⁺, wherein R is selected from the group consisting of alkyl,alkylaryl and aromatic groups. One skilled in the art recognizes thatthe counterion does not influence the function of the dye whenincorporated in a sensor. When the sensor is in a physiological fluid,the counterions equilibrate with the ions already present in the fluid.

The dyes of the invention are susceptible to quenching by electronacceptor molecules, such as viologens, they are resistant tophoto-bleaching, and are stable to photo-oxidation, hydrolysis andbiodegradation when used under conditions normally encountered inglucose sensing applications. In some embodiments, the dye is bound to apolymer through sulfonamide fimctional groups. The polymeric dyes may bewater soluble, water insoluble, organic-solvent soluble ororganic-solvent insoluble. For sensing to occur, the sensing moieties(analyte, dye and quencher) are in close physical proximity to allowinteraction, i.e., mixed on a molecular level and in equilibrium withthe species to be detected for quenching to occur.

Quenchers

As used herein, the term “quencher” refers to a compound that reducesthe emission of a fluorophore when in its presence.

In some embodiments, a quencher moiety provides glucose recognition.Such moieties comprise an aromatic boronic acid. More specifically, theboronic acid is covalently bonded to a conjugated nitrogen-containingheterocyclic aromatic bis-onium structure (e.g., a viologen) in whichthe boronic acid reacts reversibly with glucose in aqueous, organic orcombination media to form boronate esters. The extent of the reaction isrelated to glucose concentration in the medium.

Bis-onium salts are prepared from conjugated heterocyclic aromaticdinitrogen compounds. The conjugated heterocyclic aromatic dinitrogenare, e.g., dipyridlys, dipyridyl ethylenes, dipyridyl phenylenes,phenanthrolines, and diazafluorenes. It is understood that all isomersof said conjugated heterocyclic aromatic dinitrogen compounds in whichboth nitrogens can be substituted are useful in this invention.

In some embodiments, 3,3′-oBBV may be used as a quencher moiety. Thestructure of 3,3′-oBBV is:

Mono-Substituted Dyes

N-substituted mono- sulfonamide derivatives of HPTS having the genericstructure below are disclosed in the present invention:

-   -   wherein M is a counterion and R1 and R2 are individually H— or        an organic group or wherein R1 and R2 optionally comprising a        reactive group and an anionic group, preferably a sulfonate ion,        with the proviso that if one of R1 and R2 is H, the other is an        organic group and if both R1 and R2 are organic groups at least        one of R1 and R2 comprise a reactive group selected from a        polymerizable group or a coupling group, preferably a        polymerizable group. In some embodiments, R1 and R2 may be        bonded together in a cyclic structure. Polymerizable groups are        preferably ethylenically unsaturated groups including acryloyl,        methacryloyl, acrylamide, methacrylamido, styryl, and the like.        Coupling groups used to bond the dye to an existing polymer or        substrate include, but are not limited to, carboxylic acids,        aldehydes, alkynes and azides, as well as activated esters, such        as succinimides and nitrobenzoates.

Dyes with only one polymerizable group are advantageous for makinghydrogels and other sensing polymers because, in contrast to thepolymerizable HPTS derivatives in the prior art, they do not act ascrosslinkers. In addition, dye groups bonded to the polymer matrix atonly one point are hypothesized to have greater mobility in theimmobilized state thus allowing better interaction with the quencher.Interaction is further enhanced by the presence of acid groups that arefully ionized at physiological pH. Preferred dyes are mono-substitutedderivatives of HPTS wherein one sulfonate group on the pyrene ring isconverted to an N-substituted sulfonamide. The N-substituent comprises alinking group covalently bonded to an ethylenically unsaturated group,and optionally a sulfonic acid group, or salts thereof. Ethylenicallyunsaturated groups are preferably acryloyl, methacryloyl, acrylamido,methacrylamido, and styryl. Dyes with mono-substitution are alsoadvantageous because the pK_(a) of such dyes is optimized forphysiological conditions, i.e., pH 7.4.

In some embodiments, N-substituted sulfonamide derivatives are formed byreaction of a sulfonyl chloride intermediate with a primary amine,R¹—NH₂. In other embodiments, N,N-bis-substituted sulfonamidederivatives are formed by reaction with a secondary amine, R¹—NH—R²;optionally the R groups may be joined to form a cyclic secondary amine

The following scheme includes examples of structures that encompassdifferent types of mono-substituted dyes with secondary and aromaticamines:

Some HPTS dye structures used herein include:

-   1. Mono-CysMA

The structure of mono-CysMA is:

Of course, in some embodiments, substitutions other than Cys-MA on theHPTS core are consistent with aspects of the present invention, as longas the substitutions are negatively charged and have a polymerizablegroup. Either L or D stereoisomers of cysteic acid may be used.Likewise, in variations to mono-CysMA shown above, other counterionsbesides Na⁺ may be used, e.g., NBu₄ ⁺. In other variations, the sulfonicacid groups may be replaced with e.g., phosphoric, carboxylic, etc.functional groups.

The synthesis of mono-CysMA is given in Scheme 1. Reaction of HPTS with30% sulfuric acid at 150° C. for 20 min gave sodium3-hydroxypyrene-5-sulfonate 1 in 10% yield. Acetylation of 1 followed bychlorination of 2 gave intermediate 3, which is analogous to HPTS-Cl butwith only one sulfonyl chloride. The polymerizable group was attachedvia the cysteic acid moiety and reacted with 3 to give compound 4.Chlorosulfonation of 4 was achieved without affecting the polymerizablegroup and after purification on polystyrene beads gave mono-CysMA in 50%yield over two steps. The dye was characterized by ¹H NMR, HPLC, and MS.

Referring to Scheme 4, a 50-mL round bottom flask equipped with amagnetic stirring bar was charged with HPTS (9.5 mmols, 5 g) and 30%H₂SO₄ (35 mL). The mixture was heated at 150° C. for 20 min and thenallowed to cool at ambient temp for 10 min. The solution was poured into100 g of crushed ice and diluted to 200 mL with water. The mixture wasextracted with isopropyl acetate (200 mL×4) and concentrated in vacuo.The residue was mixed with silica gel (3 g) and crushed into a finepowder and dry loaded onto a Biotage 40 M cartridge. The residue waspurified via gradient elution using 5% MeOH: (5% NEt₃:CH₂Cl₂) to 15%MeOH:(5% NEt₃:CH₂Cl₂) to give the triethylamine salt of 1 (0.281 g). Thesalt was treated with 1 M HCl and extracted with isopropyl acetate andthe isopropyl acetate layer dried over MgSO₄ and concentrated in vacuoto give 1 as a brown/green foam. Synthesis of 1 was reported previouslyby E. Tietze and O. Bayer 1939 Ann 540:189-210. TLC (MeOH: CH₂Cl₂:NEt₃,2:7:1) R_(f)=0.23. ¹H NMR (500 MHz, CD₃OD) δ7.94 (t, J=7.6 Hz, 1H), 7.97(d, J=9.4 Hz, 1H), 8.02 (d, J=9.2 Hz, 1H), 8.11 (t, J=7.7 Hz, 2H), 8.25(s, 1H), 8.39 (d, J=9.1 Hz, 1H), 8.98 (d, J=9.3 Hz, 1H).

Referring to Scheme 5, a 50-mL round bottom flask equipped with amagnetic stirring bar was charged with 1 (1.7 mmols, 0.5 g), aceticanhydride (30 mL), and sodium acetate (3.4 mmols, 0.279 g). The mixturewas heated at 150° C. for 2 h and then allowed to cool to ambient temp.The solution was precipitated with ether:hexane (1:1, 50 mL) and thesolid collected onto a fritted funnel and washed with ether. The solidwas mixed with silica gel (3 g) and crushed into a fine powder and dryloaded onto a Biotage 40 M cartridge. The product was purified viagradient elution using 5% MeOH:CH₂Cl₂ to 15% MeOH: CH₂Cl₂ to give 2(0.4636 g) as a cream-colored solid. TLC (20% MeOH: CH₂C1₂) R_(f)=0.49.

Referring to Scheme 6, a 50-mL round bottom flask equipped with amagnetic stirring bar was charged with 2 (1.28 mmols, 0.463 g), CH₂Cl₂(20 mL), and oxalyl chloride (3.84 mmols, 1.92 mL of 2.0 M solution inCH₂Cl₂). DMF (0.2 mL) was added dropwise and the mixture was refluxedfor 27 h. The solution was cooled to room temp, mixed with 5 g of silicagel and filtered through a fritted fimnel. The filtrate containing 3 wasconcentrated in vacuo to ca. 5 mL, and freshly prepared 4 (1.63 mmols,0.872 g) was added along with NEt₃ (1.63 mmols, 0.227 mL). The mixturewas stirred for 13.5 h at room temperature and the precipitate thatformed was removed by filtration. The filtrate was concentrated in vacuoand loaded onto a Biotage 40M cartridge and was purified via gradientelution using 5% MeOH: (5% NEt₃:CHCl₃) to 30% MeOH: (5% NEt₃:CHCl₃). Thedesired fractions were combined and treated with 1 M HCl and extractedwith EtOAc. The EtOAc layer was dried over MgSO₄ and concentrated invacuo to give a yellow foam. The foam was treated with chlorosulfonicacid (5 mL) and the mixture was stirred for one hour at room temp. Thesolution was poured onto ice and made basic with 3M NaOH. The orangewater layer was adsorbed onto polystyrene-divinylbenzene beads (250 g)and washed with water to remove any salts. The desired product wasextracted from the beads using MeOH. The MeOH/water layer wasconcentrated in vacuo and then precipitated with acetone to give 0.1947g of mono-CysMA. ¹H NMR (500 MHz, D₂O ) δ0.80 (m, 2H), 2.12 (s, 3H),3.09 (m, 4H), 3.24 (m, 2H), 4.26 (t, J=6.8 Hz, 1H), 5.15 (s, 1H), 5.21(s, 1H), 8.19 (s, 1H), 8.54 (d, J=9.6 Hz, 1H), 8.91 (m, 3H), 9.14 (s,1H); MS (MALDI-TOF) C₂₆H₂₇N₃O₁₄S₄, MH⁺ 734.05.

A hydrogel was prepared containing mono-CysMA, as described previouslyin U.S. patent application Ser. No. 11/671,880, to evaluate thefluorescence properties (excitation (ex), emission (em) and pK_(a)) ofmono-CysMA. The excitation and emission spectra are given in FIG. 1 andare compared to tri-CysMA.

A pH study was carried out with the gel. The data is summarized in FIG.2. From this data the pK_(a)=7.2. Thus, mono-substitution allows for theincorporation of a polymerizable group and a sulfonate with minimalchange of pKa relative to HPTS (pk_(a)=7.3).

A 40% DMAA gel was prepared as previously described in U.S. patentapplication Ser. No. 11/671,880 (FIG. 3). Thus, the mono-CysMA functionsas a pH sensitive dye and as a glucose sensitive dye (in the same waythat tri-CysMA functions).

-   2. Mono-MA

The structure of mono-MA is:

The synthesis of mono-MA is given in scheme 2. Reaction of 3 withaminopropyl methacrylamide gives pyrene 7. Chlorosulfonation of 7 givesthe desired product Mono-MA. This dye is unique in that it contains onlytwo negative charges. Thus, one dye molecule can associate with onequencher to give a charge-balanced 1:1 complex

Referring to Scheme 7, mono-MA was made according to the followingmethods. A 50-mL round bottom flask equipped with a magnetic stirringbar was charged with 2 (0.591 mmols, 0.214 g), CH₂Cl₂ (10 mL), andoxalyl chloride (1.8 mmols, 0.9 mL of 2.0 M solution in CH₂Cl₂). DMF(0.1 mL) was added dropwise and the mixture was refluxed for 27 h. Thesolution was cooled to room temp, mixed with 5 g of silica gel andfiltered through a fritted funnel. The filtrate containing 3 wasconcentrated in vacuo to ca. 5 mL, and freshly prepared 6 (0.65 mmols,0.116 g) was added along with NEt₃ (0.7 mmols, 0.097 mL). The mixturewas stirred for 20 h at room temperature and the precipitate that formedwas removed by filtration. The filtrate was concentrated in vacuo andloaded onto a Biotage 40M cartridge and was purified via gradientelution using 5% MeOH: CH₂Cl₂ to 15% MeOH:CH₂Cl₂ to give a yellow solid(46 mg). The solid was treated with chlorosulfonic acid (1 mL) and themixture was stirred for one hour at room temp. The solution was pouredonto ice and made basic with 3M NaOH. The orange water layer wasadsorbed onto polystyrene-divinylbenzene beads (50 g) and washed withwater to remove any salts. The desired product was extracted from thebeads using MeOH. The MeOH/water layer was concentrated in vacuo andthen precipitated with acetone to give 8 mg of mono-MA.

Mono-MA dye was prepared and its quantum yield was compared to otherHPTS derivatives. A summary of the emission/absorbance (Em/Abs) ratiosfor HPTS, mono-MA, mono-CysMA and tri-CysMA are given in Table 1. Werefer to this as the apparent quantum yield since the actual numbers arearbitrary but can be used for comparison. FIG. 4, FIG. 5 and FIG. 6 givethe fluorescence excitation and emission spectra of mono-MA, mono-CysMAand tri-CysMA in a DMAA film, respectively at different pHs.

TABLE 1 Comparison of Dyes at 1 × 10⁻⁵ M in pH 7.4 PBS Intensity RatioDye Absorbance at max (“Quantum Yield”) HPTS 0.15654 553 3533 Mono-MA0.09003 258 2870 Mono-CysMA 0.15741 335 2128 Tri-CysMA 0.26113 351 1344

The Stem-Volmer curves for all the dyes are summarized in FIG. 7. Themono-MA is quenched most efficiently relative to the other dyes. Thisappears to be a result of the charge-charge matching between the dye andthe quencher; i.e., the dye has two negative charges and the quencherhas two negative charges.

The three polymerizable dyes were immobilized using 40% DMAA and theirpK_(a) determined via a pH study. The results are summarized in Table 2.

TABLE 2 Determination of Dye pKa Values Dye pKa HPTS 7.3 Mono-MA 6.7Mono-CysMA 7.2 Tri-CysMA 6.2

Relative to HPTS, the mono-substituted dyes have lower pK_(a)s. Withoutwishing to be bound by any particular theory, the effective pK_(a) ofeach dye appears to be the result of two structural modificationsrelative to HPTS: sulfonamide substitution on the pyrene core andanionic substitution on the linker. Dyes that are mono-substituted areadvantageous because their pK_(a)s render them more sensitive to pHchanges in the physiological range.

The dyes were tested in solution for glucose response in pH 7.4 PBS with3,3′-oBBV (FIG. 8). The dyes were also immobilized in 40% DMAA gelsusing the recipe described previously and a glucose response experimentwas carried out (FIG. 3).

Bis-Substituted Dyes

N-substituted bis-sulfonamide derivatives of HPTS having the genericstructure below are disclosed:

-   -   wherein M is a counterion and R¹ and R² are individually H— or        an organic group, or wherein R¹ and R² optionally comprise a        reactive group and an anionic group, preferably a sulfonate ion        with the proviso that if one of R¹ and R² is H, the other is an        organic group and if both R¹ and R² are organic groups at least        one of R¹ and R² comprise a reactive group selected from a        polymerizable group or a coupling group, preferably a        polymerizable group. In some embodiments, R¹ and R² may be        bonded together in a cyclic structure. Polymerizable groups are        preferably ethylenically unsaturated groups including acryloyl,        methacryloyl, acrylamide, methacrylamido, styryl, and the like.        Coupling groups used to bond the dye to an existing polymer or        substrate include, but are not limited to, carboxylic acids,        aldehydes, alkynes and azides, as well as activated esters, such        as succinimides and nitrobenzoates.

The following scheme includes examples of structures that encompassdifferent types of bis-substituted dyes with secondary and aromaticamines:

A bis-substituted fluorescent dye termed bis-CysMA having the structurebelow is disclosed in accordance with preferred embodiments of thepresent invention.

A method of making bis-cysMA is disclosed in accordance with anotherembodiment of the present invention. The method comprises the steps of:

Bis-substituted dyes serve as crosslinkers. They also may have a moreoptimal pK_(a), in comparison to tri-substituted dyes, that renders themmore sensitive to pH changes in the physiological range. In addition, itis possible to have multiple functionalities attached to the dye (e.g.,one sulfonamide may contain a polymerizable group while the other maycontain an anionic group such as a sulfonic acid.

Scheme 1 shows steps used to synthesize compound 3. Reaction of HPTSwith 30% sulfuric acid at 150° C. for 20 min gave sodium3-hydroxypyrene-5-sulfonate 1 in 10% yield. Acetylation of 1 followed bychlorination of 2 gave intermediate 3, which is analogous to our HPTS-Clbut with only one sulfonyl chloride.

Referring to Scheme 3, compound 3 is reacted with phenol in the presenceof base (e.g., pyridine, K₂CO₃, etc.) to obtain the sulfonate ester 8.Chlorosulfonation of 8 is carried out to give the bis-sulfonyl chloride9. Reaction of 9 with 4 gives 10, which upon deprotection with base,gives bis-CysMA.

Glucose Sensors

Glucose sensors of the present invention comprise a fluorophore operablycoupled to a glucose binding moiety, wherein glucose binding causes anapparent optical change in the fluophores concentration (e.g., emissionintensity). For example, glucose binding moieties such as viologensappended with boronic acid (e.g., 3,3′-oBBV) or pyridinium saltsfunctionalized with boronic acids are operably coupled to a fluorescentdye such as those described herein. The glucose binding moieties quenchthe emission intensity of the fluorescent dye, wherein the extent ofquenching is reduced upon glucose binding, resulting in an increase inemission intensity related to glucose concentration.

In some embodiments, the glucose sensor systems comprise a means forimmobilizing the sensing moieties (e.g., dye-quenchers) such that theyremain physically close enough to one another to interact (quenching).Where in vivo sensing is desired, such immobilizing means are preferablyinsoluble in an aqueous environment (e.g., intravascular), permeable toglucose, and impermeable to the sensing moieties. Typically, theimmobilizing means comprises a water-insoluble organic polymer matrix.For example, the dye-quencher may be effectively immobilized with a DMAA(N,N′-dimethylacrylamide) hydrogel matrix, which allows glucose sensingin vivo.

Typical sensor configurations include a light source adapted to generatelight at one or more excitation wavelengths, an optical fiber adapted totransmit light from the light source to a chemical indicator system(e.g., a fluorescent dye, quencher and immobilizing polymer), whereinthe indicator system is preferably disposed within the light path alonga distal region of the optical fiber, which is in contact with aphysiological fluid containing an amount of glucose (e.g., within ablood vessel), and a detector adapted to determine the emissionfluorescence at one or more emission wavelengths.

Glucose sensor chemistries, device configurations and hardware mayinclude any embodiments disclosed in co-pending U.S. patent applicationSer. Nos. 10/456,895, 11/296,898, 11/671,880, 11/782,553, 60/888,477,60/888,475, 60/917,309, 60/917,307, 60/915,372 and 60/949,145; each ofwhich is incorporated herein in its entirety by reference thereto.

Polymer Matrices for Sensors

For in vivo applications, the sensor is preferably used in a movingstream of physiological fluid, e.g., within a blood vessel, whichcontains one or more polyhydroxyl organic compounds or is implanted intissue such as muscle which contains said compounds. Therefore, it ispreferred that none of the sensing moieties escape from the sensorassembly. Thus, for use in vivo, the sensing components are part of anorganic polymer sensing assembly. Soluble dyes and quenchers can beconfined by a semi-permeable membrane that allows passage of the analytebut blocks passage of the sensing moieties. This can be realized byusing as sensing moieties soluble molecules that are substantiallylarger than the analyte molecules (molecular weight of at least twicethat of the analyte or greater than 1000 preferably greater than 5000);and employing a selective semipermeable membrane such as a dialysis oran ultrafiltration membrane with a specific molecular weight cutoffbetween the two so that the sensing moieties are quantitativelyretained.

Preferably the sensing moieties are immobilized in an insoluble polymermatrix, which is freely permeable to glucose. The polymer matrix may becomprised of organic, inorganic or combinations of polymers thereof. Thematrix may be composed of biocompatible materials. Alternatively, thematrix is coated with a second biocompatible polymer that is permeableto the analytes of interest.

One function of the polymer matrix is to hold together and immobilizethe fluorophore and quencher moieties providing an operable couplingbetween these moities, while at the same time allowing contact with theanalyte, and binding of the analyte to the boronic acid. To achieve thiseffect, the matrix is preferably insoluble in the medium, and in closeassociation with it by establishing a high surface area interfacebetween matrix and analyte solution. For example, an ultra-thin film ormicroporous support matrix may be used. Alternatively, the matrix isswellable in the analyte solution, e.g., a hydrogel matrix is used foraqueous systems. In some instances, the sensing polymers are bonded to asurface such as the surface of a light conduit, or impregnated in amicroporous membrane. In all cases, the matrix preferably does notinterfere with transport of the analyte to the binding sites so thatequilibrium can be established between the two phases. Techniques forpreparing ultra-thin films, microporous polymers, microporous sol-gels,and hydrogels are established in the art. All useful matrices aredefined as being analyte permeable.

Hydrogel polymers are preferred for embodiments of this invention. Theterm, hydrogel, as used herein refers to a polymer that swellssubstantially, but does not dissolve in water. Such hydrogels may belinear, branched, or network polymers, or polyelectrolyte complexes,with the proviso that they contain no soluble or leachable fractions.Typically, hydrogel networks are prepared by a crosslinking step, whichis performed on water-soluble polymers so that they swell but do notdissolve in aqueous media. Alternatively, the hydrogel polymers areprepared by copolymerizing a mixture of hydrophilic and crosslinkingmonomers to obtain a water swellable network polymer. Such polymers areformed either by addition or condensation polymerization, or bycombination process. In these cases, the sensing moieties areincorporated into the polymer by copolymerization using monomericderivatives in combination with network-forming monomers. Alternatively,reactive moieties are coupled to an already prepared matrix using a postpolymerization reaction. Said sensing moieties are units in the polymerchain or pendant groups attached to the chain.

The hydrogels useful in this invention may also be monolithic polymers,such as a single network to which both dye and quencher are covalentlybonded, or multi-component hydrogels. Multi-component hydrogels includeinterpenetrating networks, polyelectrolyte complexes, and various otherblends of two or more polymers to obtain a water swellable composite,which includes dispersions of a second polymer in a hydrogel matrix andalternating microlayer assemblies.

Monolithic hydrogels are typically formed by free radicalcopolymerization of a mixture of hydrophilic monomers, including but notlimited to HEMA, PEGMA, methacrylic acid, hydroxyethyl acrylate, N-vinylpyrrolidone, acrylamide, N,N′-dimethyl acrylamide, and the like; ionicmonomers include methacryloylaminopropyl trimethylammonium chloride,diallyl dimethyl ammonium chloride, vinyl benzyl trimethyl ammoniumchloride, sodium sulfopropyl methacrylate, and the like; crosslinkersinclude ethylene dimethacrylate, PEGDMA, N,N′-methylene-bis-acrylamidetrimethylolpropane triacrylate, and the like. The ratios of monomers arechosen to optimize network properties including permeability, swellingindex, and gel strength using principles well-established in the art.The concentration of dye is chosen to optimize emission intensity. Theratio of quencher to dye is adjusted to provide sufficient quenching toproduce the desired measurable signal.

Alternatively, a monolithic hydrogel may be formed by a condensationpolymerization.

Polymers that are capable of reacting with boronic acids to formboronate esters under the conditions of this method are not preferred asmatrix polymers. Such polymers have 1,2- or 1,3-dihydroxy substituents,including but not limited to cellulosic polymers, polysaccharides,polyvinyl alcohol and its copolymers and the like.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables, andappendices, as well as patents, applications, and publications, referredto above, are hereby incorporated by reference.

1. The compound

wherein R¹ and R² are independently selected from the group consistingof H, an anionic group and a reactive group, with the proviso that R¹and R² collectively comprise at least one anionic group and at least onereactive group; and wherein M is a counterion.
 2. The compound of claim1, wherein the anionic group is sulfonic acid.
 3. The compound of claim1 wherein said reactive group is an ethylenically unsaturatedpolymerizable group selected from the group consisting of acryloyl,methacryloyl, acrylamide, methacrylamido and styryl.
 4. The compound ofclaim 1 wherein said reactive group comprises a coupling group selectedfrom the group consisting of a carboxylic acid, aldehyde, alkyne, azide,activated ester, succinimide and nitrobenzoate, and wherein saidcoupling group is capable of binding the compound to a polymer orsubstrate.
 5. The compound of claim 1, wherein R¹ and R² are bondedtogether in a cyclic structure.
 6. The compound:

wherein M is a counterion.
 7. The compound:

wherein M is a counterion.
 8. The compound:

wherein M is a counterion.
 9. The compound:

wherein M is a counterion.
 10. The compound:

wherein M is a counterion.
 11. A method of making the compound of claim10, wherein M is Na, comprising the steps of:


12. The compound:

wherein M is a counterion.
 13. A method of making the compound of claim12, wherein M is Na, comprising the steps of:


14. The compound:

wherein R¹ and R² are independently selected from the group consistingof H, an anionic group and a reactive group, with the proviso that R¹and R² collectively comprise at least one anionic group and at least onereactive group; and wherein M is a counterion.
 15. The compound of claim14, wherein the anionic group is sulfonic acid.
 16. The compound ofclaim 14 wherein said reactive group is an ethylenically unsaturatedpolymerizable group selected from the group consisting of acryloyl,methacryloyl, acrylamide, methacrylamido and styryl.
 17. The compound ofclaim 14 wherein said reactive group comprises a coupling group selectedfrom the group consisting of a carboxylic acid, aldehyde, alkyne, azide,activated ester, succinimide and nitrobenzoate, and wherein saidcoupling group is capable of binding the compound to a polymer orsubstrate.
 18. The compound of claim 14, wherein R¹ and R² are bondedtogether in a cyclic structure.
 19. The compound:

wherein M is a counterion.
 20. A method of making the compound of claim19, wherein M is Na, comprising the steps of:


21. The compound:

wherein M is a counterion.
 22. The compound:

wherein M is a counterion.
 23. The compound:

wherein M is a counterion.
 24. The compound:

wherein M is a counterion.
 25. The compound of claim 1 or 14 comprisingL, D, or L and D sterioisomers of cysteic acid.
 26. A hydrogelcomprising a compound of claim 1 or
 14. 27. A glucose sensor comprisinga compound of claim 1 or claim
 14. 28. The glucose sensor of claim 27,further comprising a quencher moiety comprising boronic acid.
 29. Theglucose sensor of claim 28, wherein said quencher moiety comprisingboronic acid is 3,3′-oBBV:


30. A hydrogel comprising a glucose sensor of claim
 27. 31. The compoundof claim 10, 12 or 19, wherein M is Na.